Method for cancer targeting treatment and detection of arginine using albumin-binding arginine deiminase fusion protein

ABSTRACT

The present invention provides a pharmaceutical composition containing albumin-binding arginine deiminase (AAD) fusion protein for treating cancer or other arginine-dependent diseases. The AAD fusion protein can be purified from both soluble and insoluble fractions of crude proteins, binds to human serum albumin (HSA) or animal serum albumin and has its high activity with longer half life for efficient depletion of arginine in cancer cells. The specific activities of wild-type ADI and AAD fusion protein in the present invention are about 20 and about 19 U/mg (at physiological pH 7.4), respectively. The composition can be used alone or in combination with at least one chemotherapeutic agent to give a synergistic effect on cancer treatment and/or inhibiting metastasis. The AAD fusion protein can also be used as a component for detection and quantitative analysis of arginine in a testing kit for various samples including blood, food and analytical samples.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a reissue application of U.S. Pat. No.9,803,185, issued on Oct. 31, 2017 from U.S. patent application Ser. No.14/981,855, filed on Dec. 28, 2015, which is a continuation-in-partapplication of U.S. non-provisional patent application Ser. No.14/197,236 filed Mar. 5, 2014 and now granted under the U.S. Pat. No.9,255,262, which claims benefit from U.S. provisional patent applicationSer. No. 61/773,214 filed Mar. 6, 2013, and the disclosure disclosuresof which are incorporated herein by reference in its their entirety.

COPYRIGHT NOTICE/PERMISSION

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever. The following notice applies to the processes,experiments, and data as described below and in the drawings attachedhereto: Copyright © 2014-15, Vision Global Holdings Limited, All RightsReserved.

TECHNICAL FIELD

The present invention describes albumin-binding arginine deiminase (AAD)fusion protein that has been genetically modified to create a materialhaving high activity and long in vivo half-life. The present inventionfurther describes the designs for DNA and protein engineering forcreating different AAD fusion proteins. The AAD fusion proteins can beisolated and purified from soluble fraction and insoluble fraction(inclusion bodies) of the crude proteins. The present invention furtherrelates to albumin-binding arginine deiminase-containing pharmaceuticalcompositions for cancer targeting treatment and curingarginine-dependent diseases in humans and other animals. The AAD fusionprotein can also be used as a component in a testing kit for detectionof arginine.

BACKGROUND OF THE INVENTION

The incidence of pancreatic cancer, colon cancer, liver cancer, melanomaand cervical cancer in the worldwide population is increasing. Effectivetreatments for these diseases are urgently needed. In many types ofcancer including leukemia, melanoma, pancreatic, colon, renal cellcarcinoma, lung, prostate, breast, brain, cervical and liver cancers,the cancer cells are auxotrophic for arginine since they lack ofexpression of argininosuccinate synthetase (ASS), making these cancersexcellent targets for arginine depletion therapy.

Arginine is a semi-essential amino acid for humans and other mammals. Itcan be synthesized from citrulline via a two step process catalyzed bythe urea cycle enzymes argininosuccinate synthase (ASS) andargininosuccinate lyase (ASL). Arginine can be metabolized to ornithineby the enzyme arginase, and ornithine can be converted to citrulline byornithine carbamoyltransferase (OTC) in the mitochondria. The citrullinecan be utilized to synthesize arginine again. Normal cells usually donot require an exogenous supply of arginine for growth because of theabundant catalytic activity of ASS and ASL. In contrast, many types ofcancers do not express ASS and therefore are auxotrophic for arginine.Their growth is dependent on arginine solely obtained from bloodcirculation. Therefore, targeting circulating arginine by usingarginine-degrading enzymes is a feasible strategy to inhibitASS-negative tumor growth [Feun et al., Curr. Pharm. Des. 14:1049-1057(2008); Kuo et al., Oncotarget. 1:246-251 (2010)].

Arginine can be degraded by arginase, arginine decarboxylase, andarginine deiminase (ADI). Among them, arginine deiminase (ADI) appearsto have the highest affinity for arginine (a low K_(m) value). ADIconverts arginine to citrulline and ammonia, the metabolites of the ureacycle. Unfortunately, ADI can only be found in prokaryotes e.g.Mycoplasma sp. There are some problems associated with the isolation andpurification of ADI from prokaryotes. ADI isolated from Pseudomonasputida fails to exhibit efficacy in vivo because of its low enzymaticactivity in neutral pH. ADI produced from Escherichia coli isenzymatically inactive and subsequently requires multiple denaturationand renaturation process which raises the subsequent cost of production.

As the native ADI is found in microorganisms, it is antigenic andrapidly cleared from circulation in a patient. The native form of ADI isimmunogenic upon injection into human circulation with a short half-life(˜4 hours) and elicits neutralizing antibodies [Ensor et al., CancerRes. 62:5443-5450 (2002); Izzo et al., J. Clin. Oncol. 22:1815-1822(2004)]. These shortcomings can be remedied by pegylation. Among variousforms of pegylated ADI, ADI bound with PEG (molecular weight 20,000) viasuccinimidyl succinate (ADI-PEG 20) has been found to be an efficaciousformulation. However, the activity of ADI after pegylation is greatlydecreased on the order of 50% [Ensor et al., Cancer Res. 62:5443-5450(2002)]. The previous attempts to create pegylated ADI resulted inmaterials that are not homogenous (due to the random attachment of PEGon protein surface Lys residues) and also difficult to characterize andperform quality control during the manufacturing process. Also, PEG isvery expensive, greatly increasing the production cost. After theintravenous injection of pegylated ADI in vivo, leakage or detachment offree PEG is observed and the ADI (without PEG) can elicit theimmunogenicity problem. Therefore, there is a need for improvedcancer-treatment compositions, particularly, improved cancer-treatmentcompositions that have enhanced activity and in vivo half-life.

SUMMARY OF THE INVENTION

In the present invention, albumin-binding arginine deiminase (AAD)fusion protein has increased its activity and plasma half-life in orderto efficiently deplete arginine in cancer cells. Native ADI may be foundin microorganisms and is antigenic and rapidly cleared from circulationin a patient. The present invention constructs different AAD fusionproteins with one or two albumin-binding proteins to maintain highactivity with longer in vivo half-life (at least 5 days of argininedepletion after one injection). In the present invention, the albuminbinding protein in the AAD fusion protein product does not appear toinfluence its specific enzyme activity but instead appears to increasethe circulating half-life. The specific activities of wild-type ADI andAAD fusion protein in the present invention are about 20 and about 19U/mg (at physiological pH 7.4), respectively. In addition, the AADfusion protein of the present invention is generally more soluble inmost of the solutions than native or wild-type ADI of differentorganisms. Purification of native or wild-type ADI is also generallymore difficult than the AAD fusion protein of the present invention.

In its broadest sense, the present invention provides an albumin-bindingarginine deiminase fusion protein comprising a first portion comprisingone or two components selected from an albumin-binding domain, analbumin-binding peptide or an albumin-binding protein(s) fused to asecond portion comprising arginine deiminase to form the albumin-bindingarginine deiminase fusion protein such that the albumin-binding argininedeiminase fusion protein retains the activity of arginine deiminase andis also able to bind serum albumin.

The present invention further relates to albumin-binding argininedeiminase (AAD) fusion protein-containing pharmaceutical compositionsfor targeted cancer treatment in humans and other animals. The firstaspect of the present invention is to construct the modified AAD fusionprotein with high activity against cancer cells. The second aspect ofthe present invention is to purify AAD fusion protein with high purityfrom both soluble and insoluble fractions of the crude proteins. Thethird aspect of the present invention is to lengthen the half-life ofAAD fusion protein as it can bind to albumin very well in thecirculation. The fourth aspect of the present invention is to provide amethod of using the AAD fusion protein-containing pharmaceuticalcomposition of the present invention for treating cancer byadministering said composition to a subject in need thereof sufferingfrom various tumors, cancers or diseases associated with tumors orcancers or other arginine-dependent diseases. The fifth aspect of thepresent invention is to use AAD fusion protein as a component in atesting kit for detection of arginine.

The AAD fusion protein of the present invention is also modified toavoid dissociation into albumin-binding protein and ADI such that itbecomes more stable and has a longer half-life in circulation. ADI isfused to an albumin-binding domain/peptide/protein in AAD fusion productto extend the plasma half-life and reduce the immunogenicity of thefusion product. Albumin binding domain (ABD) is a peptide that bindsalbumin in the blood. There are different variants of ABD showingdifferent or improved human serum albumin (HSA) affinities. Differentvariants of ABD can be constructed and can be fused to ADI. Unlike thenaturally occurring ADI, this longer half-life property facilitates thedepletion of arginine efficiently in cancerous cells, cancer stem cellsand/or cancer progenitor cells.

The pharmaceutical composition containing AAD fusion protein can be usedfor intravenous (i.v.) injection (for rapid-acting dosage of medication)and intramuscular (i.m.) injection (for fairly rapid-acting andlong-lasting dosage of medication). The application of AAD fusionprotein in the present invention can be used in the treatment of variouscancers such as pancreatic cancer, leukemia, melanoma, head and neckcancer, colorectal cancer, lung cancer, breast cancer, prostate cancer,cervical cancer, liver cancer, nasopharyngeal cancer, esophageal cancerand brain cancer. The present invention is directed to AAD fusionproteins, to methods of treating cancer, to methods of treating and/orinhibiting metastasis of cancerous tissue, and to methods of curingarginine-dependent diseases.

The method of the present invention also includes using a combination ofdifferent chemotherapeutic drugs and/or radiotherapy with the AAD fusionprotein of the present invention to give a synergistic effect on cancertreatment.

In the presently claimed invention, an aspect relates to the use of theAAD fusion protein of the present invention as a component in a testingkit for detection of arginine in different samples (e.g. blood samplesfrom cancer patients, food samples, cell cultures). Said testing kitcomprises the AAD fusion protein of the present invention and a colorreagent. When a sample is incubated with the AAD fusion protein and thecolor reagent of the testing kit in appropriate assay conditions, thearginine is converted by the AAD fusion protein into citrulline. Thecolor reagent will be turned into pink color in the presence ofcitrulline. The presence of citrulline indicates the presence ofarginine in the sample and the intensity of the pink color developed inthe assay can be used to quantify the concentration of the arginine inthe sample by measuring the color intensity of the reaction mixture in aspectrophotometer. The sample can be prepared in solution form beforeusing the testing kit. The concentration of the arginine in the samplecan be expressed by the following formula:one unit of arginine deminase activity=1 μmol of arginine beingconverted to 1 μmol of citrulline per minute.In one embodiment, the AAD fusion protein of the present invention has aspecific activity of about 19 U/mg at pH 7.4 and 37° C., which iscomparable to the activity of wild-type ADI. There are many advantagesof using the AAD fusion protein of the present invention over wild-typeADI as enzyme of the testing kit, such as (1) simple production method,(2) longer shelf-life and (3) higher solubility. For wild-type ADI, itis usually insoluble and difficult for purification. For our AAD fusionprotein, it can be prepared both soluble and insoluble forms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the design approach for construction of different AADfusion proteins with one or two albumin-bindingdomain/peptide/protein(s) in three-dimensional structure. One or twoalbumin-binding domain/peptide/protein(s) can be fused to ADI to formthe AAD fusion protein. The position of albumin-bindingdomain/peptide/protein is far from the ADI active site. Thealbumin-binding domain/peptide/protein can be fused to the N-terminusor/and C-terminus of ADI. The structure in this figure is based on theMycoplasma arginini ADI structure (Protein Data Bank: 1LXY). (A) NativeADI; (B) AAD fusion protein with two ABD or ABD1; (C) AAD fusion proteinwith one ABD or ABD1 at N-terminus; (D) AAD fusion protein with one ABDor ABD1 at C-terminus.

FIG. 2 shows the sequence alignment for ADI in some bacterial speciesincluding Mycoplasma arginini (SEQ ID No. 23), Lactococcus lactis (SEQID No. 24), Bacillus cereus (SEQ ID No. 25) and Bacillus licheniformis(SEQ ID No. 26).

FIG. 3 shows the designs and amino acid sequences for different AADfusion proteins originated from Mycoplasma arginini (A to E) and AADfusion protein originated from Bacillus cereus (F).

FIG. 4 shows the creation of AAD fusion protein in two embodiments (A)and (B) by the use of intein-fusion proteins and expressed proteinligation (CBD, chitin binding domain) under the following schemes; (C)C-terminal fusion; (D) N-terminal fusion; (E) Intein-mediated proteinligation.

FIG. 5 shows the plasmid map of the expression vector constructed forproducing AAD fusion protein.

FIG. 6 shows the (A) gene map, (B) nucleotide sequence (SEQ ID No. 44)and (C) amino acid sequence (SEQ ID No. 40) of His-ABD-PolyN-ADI. (ADI:the Mycoplasma arginini ADI)

FIG. 7 shows the (A) gene map, (B) nucleotide sequence (SEQ ID No. 45)and (C) amino acid sequence (SEQ ID No. 41) of His-ABD-PolyN-bcADI.(bcADI, the Bacillus cereus ADI)

FIG. 8 shows the expression and purification of AAD fusion protein: (A)AAD is ˜90% soluble when expressed at 20° C. (lanes 2 and 3) and ˜90%insoluble (inclusion body) when expressed at 37° C. (lanes 4 and 5); (B)The purified AAD fusion protein in sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) gel: lane 1, purified AAD fusion protein(52.8 kDa); lane 2, molecular weight marker.

FIG. 9 shows the isoelectric point of AAD fusion protein in a pH 3-7 IEFprotein gel.

FIG. 10 shows the human serum albumin (HSA) binding of AAD. M: marker;Lane 1: HSA only; Lane 2: HSA:AAD ratio=1:1; Lane 3: HSA:AAD ratio=1:5;Lane 4: AAD only; Open Arrow: band of AAD, HSA, or AAD-HSA complexes.

FIG. 11 is a graph showing the dose response of AAD fusion protein onplasma arginine levels in mice. A dose of 100 μg of AAD is sufficient todeplete plasma arginine for at least 5 days.

FIG. 12 shows the effects of administration of AAD fusion protein intotal of 10 U per week and/or GEM on (A) tumor size; (B) appearance ofthe Mia-paca-2 pancreatic cancer xenograft; (C) tumor volume and (D)body weight of the mice.

FIG. 13 shows the effects of administration of AAD fusion protein intotal of 10 U per week and/or DTX on (A) tumor size; (B) appearance ofthe 22Rv1 prostate cancer xenograft and (C) tumor volume of the mice.

FIG. 14 shows the effects of administration of AAD fusion protein intotal of 10 U per week of the Jurkat leukemia xenografts on tumor volumeof the mice.

DEFINITIONS

The term “cancer stem cell” refers to the biologically distinct cellwithin the neoplastic clone that is capable of initiating and sustainingtumor growth in vivo (i.e. the cancer-initiating cell).

DETAILED DESCRIPTION OF THE INVENTION

Arginine is a semi-essential amino acid for humans and other mammals. Itcan be synthesized from citrulline via a two step process catalyzed byurea cycle enzymes argininosuccinate synthase (ASS) andargininosuccinate lyase (ASL). Arginine can be metabolized to ornithineby the enzyme arginase, and ornithine can be converted to citrulline byornithine carbamoyltransferase (OTC) in the mitochondria. The citrullinecan be utilized to synthesize arginine again. Normal cells do nottypically require an exogenous supply of arginine for growth because ofthe abundant catalytic activity of ASS and ASL. In contrast, many typesof cancers do not express ASS and are therefore auxotrophic forarginine. Their growth is solely dependent on arginine from circulation.Therefore, targeting circulating arginine by using arginine-degradingenzymes is a feasible strategy to inhibit ASS-negative tumor growth.

Arginine can be degraded by arginine deiminase (ADI). ADI convertsarginine to citrulline and ammonia, the metabolites of the urea cycle.Unfortunately, ADI can only be found in prokaryotes e.g. Mycoplasma sp.There are many problems associated with the isolation and purificationof arginine deiminase from prokaryotes. ADI isolated from Pseudomonasputida failed to exhibit efficacy in vivo because of its low enzymaticactivity in neutral pH. ADI produced from Escherichia coli isenzymatically inactive and subsequently requires multiple denaturationand renaturation process which raised the subsequent cost of production.The plasma half-life of the native form of ADI is short (˜4 hours) uponinjection into human circulation [Ensor et al., Cancer Res. 62:5443-5450(2002); Izzo et al., J. Clin. Oncol. 22:1815-1822 (2004)]. Theseshortcomings can be partially remedied by pegylation. Among variousforms of pegylated ADI, ADI bound with PEG (molecular weight 20,000) viasuccinimidyl succinate (ADI-PEG 20) has been found to be an efficaciousformulation. However, the activity of ADI after pegylation is greatlydecreased (by ˜50%) [Ensor et al., Cancer Res. 62:5443-5450 (2002); Wanget al., Bioconjug. Chem. 17:1447-1459 (2006)]. Also, the succinimidylsuccinate PEG linker can easily be hydrolyzed and detached from theprotein, causing immunogenic problems after a short period of use in thebody. Therefore, there is a need for improved cancer-treatmentcompositions, particularly, improved cancer-treatment compositions withenhanced activity.

ADI isolated from P. putida failed to exhibit efficacy in vivo becauseit had little enzyme activity at a neutral pH and was rapidly clearedfrom the circulation of experimental animals. ADI derived fromMycoplasma arginini is described, for example, by Takaku et al, Int. J.Cancer, 51:244-249 (1992), and U.S. Pat. No. 5,474,928. However, aproblem associated with the therapeutic use of such a heterologousprotein is its antigenicity. The chemical modification of ADI fromMycoplasma arginini, via a cyanuric chloride linking group, withpolyethylene glycol (PEG) was described by Takaku et al., Jpn. J. CancerRes., 84:1195-1200 (1993). However, the modified protein was toxic whenmetabolized due to the release of cyanide from the cyanuric chloridelinking group. In contrast, even for the ADI-PEG20, the PEG linker caneasily be hydrolyzed and detached from the protein, causing immunogenicproblems after a short period of use in the body. Therefore, there is aneed for compositions which degrade non-essential amino acids and whichdo not have the problems associated with the prior art.

In many types of cancer including melanoma, pancreatic, colon, leukemia,breast, prostate, renal cell carcinoma and liver cancers, cancer cellsare auxotrophic for arginine since they lack of expression ofargininosuccinate synthetase (ASS), making them excellent targets forarginine depletion therapy. In this invention, albumin-binding argininedeiminase (AAD) fusion proteins have high activity with long half-livesfor efficient depletion of arginine in cancer cells.

The size of the monomer for ADI is on the order of 45 kDa and it existsas dimer (on the order of 90 kDa) [Das et al., Structure. 12:657-667(2004)]. A design for construction of an AAD fusion protein is shown inFIG. 1. One or two albumin-binding domain/peptide/protein(s) with orwithout linker(s), SEQ ID NO: 46-49, are fused to ADI to form the AADfusion protein. It is noteworthy that the selection of one or twoparticular albumin-binding domain/peptide/protein(s) can be madedepending upon the type of cancer tissue to be targeted, the desiredsize and half-life of the resulting fusion protein, and whether a domainor entire protein is selected. Further, the selected albumin-bindingmaterial may be the same or different. That is, a protein and a peptidecan be fused, two proteins, two domains, a domain and a protein, etc.,as long as the resultant molecule retains the activity of the ADI and isalso able to bind serum albumin with neither function of one portion ofthe fusion protein being interfered with by the other portion of thefusion protein. The position of the albumin-bindingdomain/peptide/protein is far from the active site. The albumin-bindingdomain/peptide/protein can be fused to the N-terminus or/and C-terminusof ADI. There are different variants of ABD showing different orimproved human serum albumin (HSA) affinities. Different variants of ABDcan be constructed and can be fused to ADI. Some micro-organisms endowedwith ADI (for example Pseudomonas sp) cannot be used, due to theirpotential pathogenicity and pyrogenicity. The source of ADI can be from,but not limited to, different microorganisms, e.g. Mycoplasma (e.g.Mycoplasma arginini, Mycoplasma arthritidis, Mycoplasma hominis),Lactococcus (e.g. Lactococcus lactis), Pseudomonas (e.g. Pseudomonasplecoglossicida, Pseudomonas putida, Pseudomonas aeruginosa),Streptococcus (e.g. Streptococcus pyogenes, Streptococcus pneumoniae),Escherichia, Mycobacterium (e.g. Mycobacterium tuberculosis) andBacillus (e.g. Bacillus licheniformis, Bacillus cereus). It is preferredthat ADI is cloned from Mycoplasma arginini, Lactococcus lactis,Bacillus licheniformis, Bacillus cereus, thermophilic Aspergillusfumigatus or any combination thereof. Their amino acid sequences withSEQ ID (SEQ ID NO: 23-35) and the sequence alignment for some of theamino acid sequences in FIG. 2 are disclosed herein and also in theliteratures [Das et al., Structure. 12:657-667 (2004); Wang et al.,Bioconjug. Chem. 17:1447-1459 (2006); Ni et al., Appl. Microbiol.Biotechnol. 90:193-201 (2011); El-Sayed et al., Biotechnol Prog.31(2):396-405 (2015)], where the disclosure of the literatures areincorporated herein by reference in their entirety.

The design and amino acid sequence for (A) native Mycoplasma argininiADI protein (SEQ ID NO: 23), (B) different AAD fusion proteinsoriginated from the Mycoplasma arginini ADI (SEQ ID NO: 36-40) and (C)AAD fusion protein originated from the Bacillus cereus ADI (SEQ ID NO:41) are shown in FIG. 3. Different AAD fusion proteins are successfullyconstructed. A linker is inserted between the albumin-binding proteinand ADI in the AAD fusion protein in these embodiments.

On the other hand, a novel AAD fusion protein is also created by the useof intein-fusion proteins and expressed protein ligation (FIG. 4). Thenovel AAD fusion protein can be formed (1) by reacting the ADI having aN-terminal cysteine residue with a reactive thioester at C-terminus ofthe ABD, or (2) by reacting the ABD having a N-terminal cysteine residuewith a reactive thioester at C-terminus of the ADI so that the ADI andthe ABD are linked by a covalent bond. In FIG. 4E, ADI with N-terminalcysteine residue reacts with reactive thioester at the C-terminus ofABD. The thioester tag at the C-terminus of ABD, and an α-cysteine atthe N-terminus of ADI are required to facilitate protein ligation. Thesefragments are produced using a pTWIN1 vector (New England Biolabs)according to the manufacturer's manual. In particular, the gene codingfor the ABD-Intein-CBD fusion protein is synthesized and it is clonedinto the vector under the control of T7 promoter for expression in E.coli (FIG. 4C). The ABD-Intein-CBD fusion protein produced binds tochitin in a column. The amino acid sequence of ABD-Intein-CBD (SEQ IDNO: 42) is shown in FIG. 4A. After thiol-inducible cleavage and elutionfrom the column, the ABD with reactive thioester at its C-terminus isobtained (FIG. 4C). On the other hand, the gene coding for theCBD-Intein-ADI fusion protein is synthesized and cloned into the vectorunder the control of the T7 promoter for expression in E. coli (FIG.4D). The CBD-Intein-ADI fusion protein produced binds to chitin in acolumn. The amino acid sequence of the CBD-Intein-ADI (SEQ ID NO: 43) isshown in FIG. 4B. After cleavage at pH 7 and 25° C., and elution fromthe column, the ADI with α-cysteine at its N-terminus is obtained (FIG.4D). Finally, the AAD fusion protein is produced by the protein ligationreaction as shown in FIG. 4E.

Importantly, AAD fusion proteins can be produced and purified in aconvenient manner. For example, an AAD fusion protein is successfullyexpressed and purified from E. coli both in soluble fraction andinsoluble fraction, and this result is shown in FIG. 8. Furthermore,FIG. 8 shows the purified AAD fusion protein analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The size of thepurified AAD fusion protein is determined as 52.8 kDa. The isoelectricpoint of wild-type ADI is 4.7 and the purified AAD fusion protein isabout 5.2 (as shown in FIG. 9).

The pharmaceutical composition of the present invention contains AADfusion protein with high activity for depleting arginine in tumor cellsfor cancer treatment. The specific activity of the purified AAD fusionprotein is found to be similar to that of the wild-type ADI. Theinhibitory effect of the AAD fusion protein on a panel of human cancercell lines is therefore examined using MTT assay. Different cancer cellsare seeded in 96-well plates and allowed to grow for 24 h foracclimatization. The cells are then incubated with 0-10 μg/ml of AAD for72 hours. IC₅₀ is the half maximal inhibitory concentration, that is, itrepresents the concentration of AAD fusion protein that is required for50% inhibition of a cancer cell line. The IC₅₀ is a measure of theeffectiveness of a drug. The IC₅₀ of AAD fusion protein (amino acidsequence is shown in SEQ ID NO: 40, FIG. 3E) for different cancer celllines (human melanoma, A375 & SK-mel-28; human colorectal cancer,HCT116; human pancreatic cancer, Pancl & Mia-paca-2; human liver cancer,Sk-hep1; human cervical cancer, C-33A; human breast cancer, MDA-MB-231;human prostate cancer, 22Rv1; and human leukemia, Jurkat) is shown inTABLE 1.

TABLE 1 Cancer cell line IC₅₀ of AAD (argininosuccinatesynthetase-negative, ASS^(−ve)) (μg/ml) A375 (human melanoma) 0.104SK-mel-28 (human melanoma) 1.92 PancI (human pancreatic cancer) 0.043Mia-paca-2 (human pancreatic cancer) 0.010 Sk-hep1 (human livercancer) >10 C-33A (human cervical cancer) 0.058 HCT116 (human colorectalcancer) 0.211 MDA-MB-231 (human breast cancer) 0.173 22Rv1 (humanprostate cancer) 0.235 Jurkat (human leukemia) 0.379

For the albumin binding study, the present invention has demonstratedsuccessfully that the engineered AAD fusion protein can bind to humanserum albumin (HSA) or other animal serum albumin similar to HSA. FIG.10 shows that the AAD fusion protein (amino acid sequence is shown inSEQ ID NO: 40, FIG. 3E) binds to HSA readily. AAD is incubated with theindicated molar ratio of HSA for 60 min at room temperature as shown inFIG. 10, lanes 1-4. After incubation, samples are subject to nativepolyacrylamide gel (10%). Lane 2 shows a partial binding of HSA in a 1:1ratio while a complete binding of HSA is observed in a 1:5 (HSA:AAD)ratio in lane 3. At a mole ratio of 1:5 or 1:1 (i.e. lane 3 or 2 in FIG.10), the formation of the HSA-AAD complex forms (˜100-110 kDa) accordingto the construct of FIG. 1 using the linker molecule design. A band withmolecular weight ˜100 kDa representing the AAD-HSA complexes (indicatedwith an open arrowhead) is clearly observed in lane 3. It is expectedthat the circulating half-life of AAD fusion protein in the blood isincreased by the non-covalent HSA-AAD complex formation. Therefore, along-lasting version of AAD fusion protein has been successfullycreated.

No commercial products show high efficacy when compared to the AADfusion protein-containing pharmaceutical composition prepared in thisinvention. For uses in cancer treatment, the AAD fusionprotein-containing pharmaceutical composition of the present inventionserves as an anticancer agent to deplete the arginine in tumor tissues.AAD fusion protein is a good candidate to be used in combination withother molecular targeting or cytotoxic agents.

The AAD fusion protein in the present invention can also be used as acomponent in a testing kit for detection of arginine in differentsamples. AAD has a small Km value; it indicates the high affinity forthe substrate (arginine). Therefore, the rate will approach the maximumreaction rate more quickly. The AAD fusion protein can be used totesting arginine level (1) in cancer patients, (2) in a food sample, and(3) in cell culture.

EXAMPLES

The following examples are provided by way of describing specificembodiments of this invention without intending to limit the scope ofthis invention in any way.

Several of the Examples below relate to methods of making analbumin-binding arginine deiminase fusion protein. Various techniquescan be used including cloning and intein-mediated protein ligation. Asused herein, the term “cloning” is broadly used and comprisesconstructing a fusion gene coding for the albumin-binding argininedeiminase fusion protein, inserting the fusion gene into a vector,inserting the vector into a host organism and expressing a protein thatincludes an albumin-binding arginine deiminase fusion protein. Numerousvariants on this technique can be performed and still fall within thecloning contemplated by the present invention.

Example 1 Construction of the Gene Coding for Albumin-BindingDomain/Peptide/Protein (ABD)

The gene coding for ABD is constructed by two rounds of PCR. In thefirst round, the PCR reaction mixture (total volume of 25 μl) containsthe following materials:

1×iProof PCR buffer (Bio-Rad)

50 μM dNTP mixture

0.5 unit of iProof DNA Polymerase (Bio-Rad)

10 nM of each of the following oligos

ABD-F1 forward primer (SEQ ID NO: 01):5′-CATGATGCGAATTCCTTAGCTGAAGCTAAAGTCTT AGCTAACAGAGAACT-3′ABD-R2 reverse primer (SEQ ID NO: 02):5′-TAGTCACTTACTCCATATTTGTCAAGTTCTCTGTT AGCTAAGACTTTAGC-3′ABD-F3 forward primer (SEQ ID NO: 03):5′-GAACTTGACAAATATGGAGTAAGTGACTATTACAA GAACCTAATCAACAA-3′ABD-R4 reverse primer (SEQ ID NO: 04):5′-TACACCTTCAACAGTTTTGGCATTGTTGATTAGGT TCTTGTAATAGTCAC-3′ABD-F5 forward primer (SEQ ID NO: 05):5′-GCCAAAACTGTTGAAGGTGTAAAAGCACTGATAGA TGAAATTTTAGCTGC-3′ABD-R6 reverse primer (SEQ ID NO: 06):5′-AGCTACGATAAGCTTAAGGTAATGCAGCTAAAATT TCATCTATCAGTG-3′The following PCR program is used:98° C. 30 s; 20 cycles of {98° C. 10 s, 50° C. 20 s, 72° C. 20 s}

In the second round of PCR, the PCR mixture (total volume of 50 μl)contains the following materials:

1×iProof PCR buffer (Bio-Rad);

50 μM dNTP mixture;

1 μl of PCR reactant as DNA template from the first round;

1 unit of iProof DNA Polymerase (Bio-Rad);

200 nM of each of the following oligos:

ABD-F7 forward primer (SEQ ID NO: 07):5′-CATGATGCGAATTCCTTAGCTGAAGCTAAAGTCTT AGCTAACAGAGAACT-3′ABD-R8 reverse primer (SEQ ID NO: 08):5′-AGCTACGATAAGCTTAAGGTAATGCAGCTAAAATT TCATCTATCAGTG-3′

The following PCR program is used:

98° C. 30 s; 35 cycles of {98° C. 10 s, 60° C. 20 s, 72° C. 20 s}; 72°C. 5 min

A PCR product containing the DNA sequence of ABD (169 bp) is obtainedand purified by Qiagen DNA Gel Extraction Kit for cloning purpose.

Example 2A Construction of the Fusion Gene Coding for the AAD FusionProtein

In the first PCR, the PCR mixture (total volume of 50 μl) contains thefollowing materials:

1×iProof PCR buffer (Bio-Rad);

50 μM dNTP mixture;

25 ng of Mycoplasma arginini genomic DNA;

1 unit of iProof DNA Polymerase (Bio-Rad);

200 nM of each of the following oligos:

ADINde-F forward primer (SEQ ID NO: 09):5′-ATCGATCGATGTCTGTATTTGACAGTAAATTTAAAG G-3′ADIhis-R reverse primer (SEQ ID NO: 10):5′-AGCTAAGGAATTCGCATCATGATGGTGATGGTGGTGG CTACCCCACTTAAC-3′The following PCR program is used:98° C. 1 min; 35 cycles of {98° C. 10 s, 50° C. 20 s, 72° C. 40 s}; 72°C. 5 minA PCR product of 1280 bp long is obtained and purified by Qiagen DNA GelExtraction Kit. After that, the second PCR is performed. The PCR mixture(total volume of 50 μl) contains the following materials:

1×iProof PCR buffer (Bio-Rad);

50 μM dNTP mixture;

10 ng of the 1280 bp PCR product;

10 ng of the 169 bp PCR product;

1 unit of iProof DNA Polymerase (Bio-Rad);

200 nM of each of the following oligos:

ADINde-F forward primer (SEQ ID NO: 11):5′-ATCGATCGATGTCTGTATTTGACAGTAAATTTAAAG G-3′ABD-R10 reverse primer (SEQ ID NO: 12):5′-AGCTACGATAAGCTTAAGGTAATGCAGCTAAAATTT CATCTATCAGTG-3′The following PCR program is used:98° C. 1 min; 35 cycles of {98° C. 10 s, 50° C. 20 s, 72° C. 45 s}; 72°C. 5 min

A PCR product of 1428 bp is obtained and purified by Qiagen DNA GelExtraction Kit. Then it is digested with restriction enzymes NdeI andHindIII, and ligated to plasmid pREST A (Invitrogen) that is predigestedwith the same enzymes. The ligation product is then transformed into E.coli BL21 (DE3) cells. The sequence of the constructed fusion gene isconfirmed by DNA sequencing.

Example 2B Cloning of His-ABD-PolyN-ADI

The construction of His-ABD-PolyN-ADI (SEQ ID NO: 40, in FIG. 3E) isdone by two steps of overlapping PCR, the PCR fragment obtained from thelast step is inserted into the vector pET3a between the NdeI and BamHIsites. The gene map, nucleotide sequence and amino acid sequence ofHis-ABD-PolyN-ADI are shown in FIG. 6.

Primers involved in construction of His-ABD-PolyN-ADI:

hisABDNde-F forward primer (SEQ ID NO: 13):5′-GGAGATATACATATGCATCATCACCATCACCATGATGAAG CCGTGGATG-3′ABDnn-R1 reverse primer (SEQ ID NO: 14):5′-TTGTTATTATTGTTGTTACTACCCGAAGGTAATGCAGCTA AAATTTCATC-3′ABDn-R2 reverse primer (SEQ ID NO: 15):5′-AGAACCGCCGCTACCATTGTTATTATTGTTGTTACTACCC GA-3′ADln-F forward primer (SEQ ID NO: 16):5′-AATAATAACAATGGTAGCGGCGGTTCTGTATTTGACAGTA AATTTAAAGG-3′ADIBam-R reverse primer (SEQ ID NO: 17):5′-TAGATCAATGGATCCTTACCACTTAACATCTTTACGTGAT AAAG-3′

In the first round of PCR, 50 μl of reaction volume containing the knownconcentration of components are prepared in two PCR tubes. In each ofthe tubes, dNTP, iProof buffer (BIO-RAD), iProof DNA polymerase(BIO-RAD), primers and DNA template are mixed and added up to 50 μl byddH₂O. The DNA template used in the reaction is a pET3a vectorcontaining the gene of ADI from Mycoplasma arginini with a removal of aninternal NdeI site mutation without altering the protein sequence of theADI gene.

The two reaction tubes contain the primer mixtures of (A) 10 pmolhisABDNde-F (SEQ ID NO: 13), 0.5 pmol ABDnn-R1 (SEQ ID NO: 14) and 10pmol ABDn-R2 (SEQ ID NO: 15); and (B) 10 pmol ADIn-F (SEQ ID NO: 16) and10 pmol ADIBam-R (SEQ ID NO: 17), respectively.

The PCR program is set according to the recommended steps in the manualwith an annealing and extension temperature (time) at 50° C. (20 s) and72° C. (40 s), respectively. The two products generated by PCR with thesize of 237 bp and 1278 bp. The products are extracted and applied astemplate for the next round of PCR.

In the second overlapping step, the reaction mixture is prepared in asimilar way to the first round except the template used was the mixtureof 1 pmol of the 237 bp PCR product and 1 pmol of the 1278 bp PCRproduct from the first round PCR. Primers used are changed to 10 pmolhisABDNde-F (SEQ ID NO: 13) and 10 pmol ADIBam-R (SEQ ID NO: 17).

The annealing and extension temperature (time) are 50° C. (20 s) and 72°C. (60 s), respectively. A PCR product with the size of 1484 bp isgenerated from the reaction. The PCR product is purified and digestedwith NdeI and BamHI and then ligated into the pre-digested pET3aplasmid. The ligated product is then transformed into E. coli BL21 (DE3)for the production of recombinant protein.

Example 2C Cloning of His-ABD-PolyN-bcADI

The construction of His-ABD-PolyN-bcADI (SEQ ID NO: 41, in FIG. 3F) isdone by two steps of overlapping PCR, the PCR fragment obtained from thelast step is inserted into the vector pET3a between the NdeI and BamHIsites. The gene map, nucleotide sequence and amino acid sequence ofHis-ABD-PolyN-bcADI are shown in FIG. 7.

Primers involved in construction of His-ABD-PolyN-bcADI:

hisABDNde-F2 forward primer (SEQ ID NO: 18):5′-GGAGATATACATATGCATCATCACCATCACCATGATGAAGC CGTGGATG-3′bcABDnn-R1 reverse primer (SEQ ID NO: 19):5′-TTGTTATTATTGTTGTTACTACCCGAAGGTAATGCAGCTAA AATTTCATC-3′bcABDn-R2 reverse primer (SEQ ID NO: 20):5′-TTTACCGCCGCTACCATTGTTATTATTGTTGTTACTACCCG A-3′bcADln-F forward primer (SEQ ID NO: 21):5′-AATAATAACAATGGTAGCGGCGGTAAACATCCGATACATGT TACTTCAGA-3′bcADIBam-R reverse primer (SEQ ID NO: 22):5′-TAGATCAATGGATCCCTAAATATCTTTACGAACAATTGGCA TAC-3′

In the first round of PCR, 50 μl of reaction volume containing the knownconcentration of components are prepared in two PCR tubes. In each ofthe tubes, dNTP, iProof buffer (BIO-RAD), iProof DNA polymerase(BIO-RAD), primers and DNA template are mixed and added up to 50 μl byddH₂O. The DNA template used in the reaction is a pET3a vectorcontaining the gene of ADI from Bacillius cereus with a removal of aninternal NdeI site mutation without altering the protein sequence of theADI gene.

The two reaction tubes contain the primer mixtures of (A) 10 pmolhisABDNde-F2 (SEQ ID NO: 18), 0.5 pmol bcABDnn-R1 (SEQ ID NO: 19) and 10pmol bcABDn-R2 (SEQ ID NO: 20); and (B) 10 pmol bcADIn-F (SEQ ID NO: 21)and 10 pmol bcADIBam-R (SEQ ID NO: 22), respectively. The PCR program isset according to the recommended steps in the manual with an annealingand extension temperature (time) at 50° C. (20 s) and 72° C. (40 s),respectively. The two products are generated by PCR with the size of 237bp and 1250 bp. The products are extracted and applied as template forthe next round of PCR.

In the second overlapping step, the reaction mixture is prepared in asimilar way to the first round except the template used is the mixtureof 1 pmol of the 237 bp PCR product and 1 pmol of the 1250 bp PCRproduct from the first round PCR. Primers used are changed to 10 pmolhisABDNde-F2 (SEQ ID NO: 18) and 10 pmol bcADIBam-R (SEQ ID NO: 22).

The annealing and extension temperature (time) are 50° C. (20 s) and 72°C. (60 s), respectively. A PCR product with the size of 1512 bp isgenerated from the reaction. The PCR product is purified and digestedwith NdeI and BamHI and then ligated into the pre-digested pET3aplasmid. The ligated product is then transformed into E. coli BL21 (DE3)for the production of recombinant protein.

Example 3 Expression and Purification of the AAD Fusion Protein

(3a) Expression of the AAD Fusion Protein by Shake-Flask Method

For preparing the seed culture, the strain E. coli BL21 (DE3) carryingthe plasmid encoding the AAD fusion protein (FIG. 5) is cultured in 5 mlof 2×TY medium, 30° C., 250 rpm, overnight. The overnight seed culture(2.5 ml) is added to 250 ml of 2×TY, 37° C., 250 rpm, 2.5 h (untilOD₆₀₀≈0.6-0.7). When the OD₆₀₀ reached, IPTG is added to the culture(0.2 mM final concentration). The growth is continued for 22 more hoursat 20° C. and then the cells are collected by centrifugation. The cellpellet is resuspended in 25 ml of 10 mM sodium phosphate buffer, pH 7.4.The cells are lysed by sonication.

(3b) Expression of the AAD Fusion Protein by Fermentation Method

For the seed culture, the aliquot of bacteria stock is inoculated into50 ml of seeding medium (containing 1.5 g of yeast extract and 0.25 g ofNaCl) with ampicillin and grown at 30° C. for 16 hr with continuousshaking at 250 rpm. The seed culture is then added to 1.25 L of medium(pH 7.4, containing yeast extract, tryptone, Na₂HPO₄, KH₂PO₄, (NH₄)₂SO₄,glycerol, glucose, MgSO₄.7H₂O, Thiamine-HCl and CaCl₂) supplement withtrace element in the BIOSTAT fermentor system and grown at 28° C. Untilthe OD₆₀₀ of the culture reaches ˜20, IPTG is added to a finalconcentration of 0.2 mM. The culture is further incubated for 16 hr.During incubation, 500 ml of feeding medium (pH 7.4, containing yeastextract, tryptone, NH₄Cl, (NH₄)₂SO₄, glycerol and MgSO₄.7H₂O) supplementwith trace element were applied at 0.5 ml/min. Aeration is regulated inorder to maintain 20% of air saturation by varying the speed of stirringfrom 500 rpm to 2000 rpm. The bacteria are harvested by centrifugation.The cells are lysed by sonication or high pressure homogenizer.

(3c) Purification of the AAD Fusion Protein

The soluble portion is collected after centrifugation. The fusionprotein (containing a His tag) is then purified by nickel affinitychromatography. TABLE 2 shows that cultivation temperature is animportant factor in affecting the solubility of AAD fusion protein(amino acid sequence is shown in SEQ ID NO: 40, FIG. 3E) obtained fromthe expression host.

For isolating the soluble fraction of AAD fusion protein, the cellpellet is resuspended in 25 ml of 10 mM sodium phosphate buffer, pH 7.4.The cells are lysed by sonication or high pressure homogenizer. Thesoluble portion is collected after centrifugation. The AAD fusionprotein (contains a His tag or without His tag) is then purified bynickel affinity chromatography and/or ion-exchange columns.

For isolating the insoluble fraction of AAD fusion protein, the cellpellet is resuspended in 25 ml of 20 mM Tris-HCl, pH 7.4, 1% TRITONX-100. The cells are lysed by sonication. The insoluble portion(inclusion bodies) is collected by centrifugation. The protein isunfolded by resuspending in 10 ml of 20 mM Tris-HCl, pH 7.4, 6 MGuanidine HCl, and vortexed until it becomes soluble. The protein isrefolded by adding the unfolded protein solution drop by drop into afast stiffing solution of 100 ml of 20 mM Sodium phosphate buffer, pH7.4. The insoluble materials are removed by centrifugation. Salting outof the protein is performed by adding solid ammonium sulphate powderinto the supernatant to achieve 70% saturation. The insoluble portion iscollected by centrifugation and it is resuspended in 10 ml of 20 mMsodium phosphate buffer. The AAD fusion protein (contains a His tag orwithout His tag) is then purified by nickel affinity chromatographyand/or ion-exchange columns.

TABLE 2 AAD 1 2 3 Cultivation 30 20 37 temperature (° C.) Yield (mg)/~0.66 ~12.0 ~7.0 250 ml culture solubility 50% soluble 90% soluble 90%inclusion body IC₅₀ (μg/ml) on 0.10 0.68 0.23 A375 cells“90% inclusion body” means 90% of the AAD fusion protein produced in thebacterial cells are not soluble.

The yield and the enzyme activity of AAD fusion protein from shake-flaskmethod and fermentation method are shown in TABLE 3.

TABLE 3 Activity AAD Yield (mg/L) (U/mg) Shake-flask method ~10 ~9Fermentation method ~42 ~19

Example 4 Enzyme Activity Assay and Enzyme Kinetics for AAD FusionProtein

To determine the enzyme activity for wild-type ADI and AAD fusionprotein in the present invention, the diacetyl monoxime(DAM)-thiosemicarbazide (TSC) assay for citrulline detection is used.The reaction is shown below.L-Arginine_(argininedeiminase(ADI)orAADfusion) >L-Citrulline+Ammonia

This assay is run by adding sample to a color reagent, which is made bymixing acidic ferric chloride solution with DAM-TSC solution. Briefly,enzyme is incubated with 20 mM arginine, 10 mM sodium phosphate pH 7.4for 5 min at 37° C. The reaction mixture is heated at 100° C. for 5 mMto develop the color and read at 540 nm (light path=1 cm). A standardcurve is constructed using various concentrations of citrulline. Oneunit of the ADI native enzyme is the amount of enzyme activity thatconverts 1 μmol of arginine to 1 μmol of citrulline per minute at 37° C.under the assay conditions. The specific activities of wild-type ADI,pegylated ADI (Ensor et al., Cancer Res. 62:5443-5450, 2002) and AADfusion protein in the present invention are about 20, 15 and 19 U/mg (atpH 7.4, physiological pH), respectively. The specific activities forwild-type ADI and AAD fusion protein at different pH values (in a rangefrom pH 5.5 to 9.5) are also determined, and the optimum pH is at 6.5.Therefore, the results indicate that AAD fusion protein depletesarginine efficiently which is even better than pegylated ADI, as thefusion with albumin-binding protein does not affect enzyme activity ofADI.

The Michaelis constant K_(m) is the substrate concentration at which thereaction rate is at half-maximum, and is an inverse measure of thesubstrate's affinity for the enzyme. A small K_(m) indicates highaffinity for the substrate, and it means that the rate will approach themaximum reaction rate more quickly. For determination of the enzymekinetics or K_(m) value, the activity of wild-type ADI and AAD fusionprotein are measured under different concentration of substrate arginine(2000 μM, 1000 μM, 500 μM, 250 μM, 125 μM, 62.5 μM) at pH 7.4. Themeasured K_(m) values of the AAD fusion protein shown in FIG. 3E (SEQ IDNO: 40, ADI protein is originated from Mycoplasma arginini) and AADfusion protein shown in FIG. 3F (SEQ ID NO: 41, ADI protein isoriginated from Bacillus cereus) are 0.0041 mM and 0.132 mMrespectively. The results suggest that the fusion to ABD did not affectthe binding affinity of the different AAD fusion proteins to arginine.

Example 5 Cell Proliferation Assay and In Vitro Efficacy of AAD FusionProtein on Cancer Cell Lines

Culture medium DMEM is used to grow the human melanoma A375 & SK-mel-28and pancreatic cancer Pancl & Mia-paca-2 cell lines. The EMEM medium isused to culture the human liver cancer SK-hep, cervical cancer C-33A andcolorectal cancer HCT116 cell lines. The RPMI-1640 medium is used toculture the human breast cancer MDA-MB-231, prostate cancer 22Rv1 andleukemia Jurkat cell lines. Cancer cells (2-5×10³) in 100 μl culturemedium are seeded to the wells of 96-well plates and incubated for 24hours. The culture medium is replaced with medium containing 0-10 μg/mlof AAD fusion protein. The plates are incubated for an additional 3 daysat 37° C. in an atmosphere of 95% air/5% CO₂. MTT assay is performed toestimate the number of viable cells in the culture according tomanufacturer's instructions. The amount of enzyme needed to achieve 50%inhibition of cell growth is defined as IC₅₀.

As shown in TABLE 1, the results indicate that AAD fusion proteindepletes arginine efficiently and inhibits the growth of various typesof human cancer cell lines in in vitro tissue culture studies. Forexample, human melanoma, human breast cancer, human colorectal cancer,human pancreatic cancer, human liver cancer, human prostate cancer,human leukemia and human cervical cancer, all have low values of IC₅₀(see TABLE 1), as these cancer types are all inhibited by AAD fusionprotein readily. As determined from the in vitro_data, AAD fusionprotein would inhibit all cancer types that are arginine-dependent, forexample, the argininosuccinate synthetase-negative (ASS^(−ve)) cancers.

Example 6 In Vivo Half-Life Determination of AAD Fusion Protein

Balb/c mice (5-7 weeks) are used in this study and they are allowed toacclimatize for a week before the experiment. Mice (n=3) are separatedinto four groups and injected with 0, 100, 500 or 1000 μg of AAD fusionprotein (SEQ ID NO: 40, FIG. 3E) in 100 μl PBS intraperitoneally,respectively. Blood of each mouse is collected at 0 h and Day 1-7. Seraare obtained after centrifugation. The sera are then deproteinised andanalyzed by amino acid analyzer for arginine.

As shown in FIG. 11, AAD fusion protein (SEQ ID NO: 40, FIG. 3E), evenat the lowest dosage of 100 μg, depletes plasma arginine efficiently atDay 1, 3 and 5, suggesting that AAD can deplete arginine in vivoefficiently for at least 5 days. The arginine level returns to normalgradually at Day 6 and Day 7 in all treatment groups.

Example 7 In Vivo Efficacy of Weekly Administration of AAD FusionProtein on HCT 116 Colorectal Cancer Cell Xenografts

Nude balb/c mice (5-7 weeks) are used in this study and they are allowedto acclimatize for a week before the experiment. Mice are inoculatedsubcutaneously with 2×10⁶ cancer cells in 100 μl of fresh culturemedium. Ten days later, the mice are randomly separated into control andtreatment group. Control group receives 100 μl PBS and treatment groupreceives 100 μl AAD fusion protein (amino acid sequence is shown in SEQID NO: 40, FIG. 3E) intraperitoneally weekly. Tumor size is measured bycaliper and tumor volume is calculated using formula: (length×width²)/2.Blood draw are obtained at Day 5 after each treatment for plasmameasurement of arginine.

Example 8 Comparison in In Vivo Efficacy Between Weekly and BiweeklyBased Administration of AAD Fusion Protein on HCT 116 Colorectal CancerCell Xenograft

Nude balb/c mice (4-6 weeks) are used and they are allowed toacclimatize for a week before the treatment. Mice are inoculatedsubcutaneously with 2×10⁶ cancer cells in 100 μl PBS. Ten days later,the mice are randomly separated into control and treatment groups.Control group receives 100 μl PBS and treatment group receives 200 μgAAD fusion protein (amino acid sequence is shown in SEQ ID NO: 40, FIG.3E) in 100 μl PBS intraperitoneally weekly or biweekly. Tumor size ismeasured by caliper and tumor volume is calculated using formula:(length×width²)/2. In the weekly treatment group, blood draw areobtained at Day 0 and Day 5 of each treatment for plasma measurement ofarginine. In the biweekly treatment group, blood draw are obtained justbefore the first treatment every week for plasma measurement ofarginine. After the mice are sacrificed by end of the time course, thetumors are excised and weighed. The plasma arginine levels over the timecourse in the weekly treatment group are measured.

At Day 5, Day 12 and Day 19, the plasma arginine level is significantlydecreased respectively after the weekly administration of the AAD fusionprotein. Comparing the plasma arginine levels between Day 0, Day 7 andDay 14, the levels at Day 7 and Day 14 are relatively lower than that atDay 0, revealing that weekly administration of AAD fusion protein candecrease the overall plasma arginine levels over the time course. Thetumor size in the weekly treatment group is lower than that in controlat the end of the time course, revealing that the weekly administrationof AAD fusion protein can reduce the tumor size of the xenograft in thedisease mouse model. The difference in the tumor size between controland weekly treatment group is about 20% at Day 30.

The reduction in plasma arginine level is more significant in thebiweekly treatment group than that in the weekly treatment group. Thebiweekly administration of AAD fusion protein does not affect the bodyweight of the mice over a 35-day time course as compared to the control.In conclusion, biweekly administration of AAD fusion protein in 400 μgper week (16 mg/kg/week/mouse) is more effective in completelysuppressing plasma arginine level than weekly administration. Using theconversion of animal doses to human equivalent doses (HED) based on bodysurface area mentioned in “Guidance for Industry andReviewers—Estimating the safe starting dose in clinical trials fortherapeutics in adult healthy volunteers (2002)”, human dose of the AADfusion protein is about 1.3 mg/kg/week.

Example 9 In Vivo Efficacy of AAD Fusion Protein on Mia-Paca-2Pancreatic Cancer Cell Xenografts

Nude balb/c mice (4-6 weeks) are used and they are allowed toacclimatize for a week before the treatment. Mice are inoculatedsubcutaneously with 2×10⁶ cancer cells in 100 μl of 1:1 PBS:Matrigel.Matrigel is used to augment the growth of tumors. Two weeks later, themice are randomly separated into four groups of 4 animals in each group.Mice are intraperitoneally administered with PBS (control), AAD (5 U;twice a week), Gemcitabine, Gem (100 mg/kg; once a week) or AAD+Gem (acombination of both AAD and Gem) in 200 μl PBS. Tumor size is measuredby caliper and tumor volume is calculated using formula:(length×width²)/2. Body weight is measured every week. After the miceare sacrificed by end of the time course, the tumors are excised andweighed. The tumor size in all treatment groups is significantly lowerthan that in control at the end of the time course, revealing that theadministration of AAD fusion protein can reduce the tumor size of thexenograft in the disease mouse model (shown in FIG. 12A, 12B, 12C). Thedifference in the tumor size between control and AAD fusionprotein-treatment group is about 60% at Day 28. The treatment groups donot affect the body weight of the mice over a 28-day time course ascompared to the control (shown in FIG. 12D).

Example 10 In Vivo Efficacy of AAD Fusion Protein on 22Rv1 ProstateCancer Cell Xenografts

Nude balb/c mice (4-6 weeks) are used and they are allowed toacclimatize for a week before the treatment. Mice are inoculatedsubcutaneously with 3×10⁶ cancer cells in 100 μl PBS. Two weeks later,the mice are randomly separated into four groups of 5 animals in eachgroup. Mice are intraperitoneally administered with PBS (control), AAD(5 U; twice a week), Docetaxel, DTX (10 mg/kg; once a week) or AAD+DTX(a combination of both AAD and DTX) in 200 ul PBS. Tumor size ismeasured by caliper and tumor volume is calculated using formula:(length×width²)/2. Body weight is measured every week. After the miceare sacrificed by end of the time course, the tumors are excised andweighed.

In FIG. 13A, the tumor size in all treatment groups is significantlylower than that in control at the end of the time course, revealing thatthe administration of AAD fusion protein or docetaxel can reduce thetumor size of the xenograft in the disease mouse model. The differencein the tumor size between control and AAD fusion protein- orDTX-treatment groups are about 55% and 54% at Day 22, respectively.Besides, the combination of AAD fusion protein and DTX-treatment grouphas a synergistic effect on tumor growth inhibition. Tumor growth of thecombination treatment group is inhibited by about 94% when compared tothat of the control group. FIG. 13B shows the appearance of the tumortissues excised from xenografts at Day 22. AAD treatment does not affectthe body weight of the mice over a 28-day time course.

Example 11 In Vivo Efficacy of AAD Fusion Protein on Jurkat LeukemiaXenografts

Nude balb/c mice (4-6 weeks) are used and they are allowed toacclimatize for a week before the treatment. Mice are inoculatedsubcutaneously with 5×10⁶ cancer cells in 100 μl of 1:1 PBS:Matrigel.Matrigel is used to augment the growth of tumors. Since the take rate ofthis cancer cell line is relatively low, when certain tumor xenograftreaches a suitable size, the tumor is excised and cut into variouspieces (˜10 mm³), which are further transplanted to the back of anothergroup of mice subcutaneously. Ten days later, the mice are randomlyseparated into two groups of 8 animals in each group. Mice areintraperitoneally administered with PBS (control) or AAD (5 U; twice aweek) in 200 μl PBS. Tumor size is measured by caliper and tumor volumeis calculated using the following formula: (length×width²)/2. Bodyweight is measured every week. At Day 28, the AAD fusion proteinsignificantly inhibits the tumor growth when comparing to the controlgroup (FIG. 14). The difference in tumor size between AAD fusionprotein-treated and control groups becomes more significant at Day 30.

Example 12 Using AAD Fusion Protein in a Testing Kit

AAD fusion protein of the present invention has a small K_(m),indicating the high affinity for the substrate (arginine). Therefore,the rate approaches the maximum reaction rate more quickly. To determinethe concentration of arginine in a sample, AAD fusion protein in thepresent invention, the diacetyl monoxime (DAM)-thiosemicarbazide (TSC)assay for citrulline detection is used. The reaction is as follows:L-Arginine (of unknown concentration) is converted by AAD fusion proteinto form L-Citrulline and Ammonia.

This assay is run by adding sample to a color reagent, which is made bymixing acidic ferric chloride solution with DAM-TSC solution. Briefly,arginine (in the sample, of unknown concentration) is incubated with2-20 ng of the AAD fusion protein, and 10 mM sodium phosphate pH 7.4 for5 mM at 37° C. The reaction mixture is heated at 100° C. for 5 mM todevelop into pink color and read at 540 nm (light path=1 cm). A standardcurve is constructed using various concentrations of citrulline. Oneunit of the AAD is the amount of enzyme activity that converts 1 μmol ofarginine to 1 μmol of citrulline per minute at 37° C. under the assayconditions. This testing kit is very useful for the followingapplications:

Example 12A Testing Arginine Level in Cancer Patients

For a cancer patient (human or animal), after treated with an argininedepleting drug, the arginine level (concentration) in blood should bevery low or zero. A blood sample can be taken from the patient and thentested with this new testing kit based on the AAD fusion protein. Aftercomparing to the standard curve (generated from standard solutionplotted from known arginine concentration solutions), the exact arginineconcentration of this blood sample of the patient can be measured.Therefore, this data helps monitor the progress of an arginine depletiontreatment method. If the arginine level is too high (e.g. 200micro-molar), more arginine depleting drug can be used on the patient tofurther keep the arginine at a lower or undetectable level so that thetumor can be inhibited by the systemic arginine depletion in the cancerpatient.

Example 12B Testing Arginine Level in a Food Sample

For industrial or food manufacturing purposes, the arginine (amino acid)level of a particular food material or intermediate during the foodprocessing step might need to be monitored and measured. This argininetesting kit by using the AAD fusion protein of the present invention canmeasure the exact arginine concentration of a food sample prepared insolution form in the laboratory. This testing kit can also be used inhigh throughput manner and applicable for industrial scale and massproduction.

Example 12C Testing Arginine Level in Cell Culture

Nitric oxide (NO) is an important signaling molecule in cells and in thebody. For many research projects on nitric oxide (NO) and cell culturestudies, a lot of time a scientist would need to measure the amount orconcentration of arginine (which is a substrate for making NO). Thisarginine testing kit by using the AAD fusion protein of the presentinvention can also be used for measuring the exact arginineconcentration of any laboratory samples and/or analytical samples (e.g.for NO and cell culture studies).

What is claimed is:
 1. A method of treating a cancer or inhibitingarginine-dependent tumor growth in a subject comprising administering analbumin-binding arginine deiminase fusion protein to the patient weeklyor biweekly to reduce the availability of circulating arginine, whereinsaid albumin-binding arginine deiminase fusion protein comprises a firstportion comprising one or two albumin-binding domain(s) fused to asecond portion comprising arginine deiminase to form the albumin-bindingarginine deiminase fusion protein, and one or more linker molecules; thefirst portion being positioned far from active site of the secondportion by said linker molecule such that the albumin-binding argininedeiminase fusion protein retains the activity of arginine deiminase andbinds serum albumin with neither function of one portion of the fusionprotein being interfered with by the other portion of the fusionprotein, wherein said albumin-binding arginine deiminase fusion proteincomprises a sequence selected from SEQ ID NO: 36, 37, 38, 39, 40 or 41,and wherein said cancer consists essentially of is selected from thegroup consisting of pancreatic cancer, leukemia, melanoma, head and neckcancer, colorectal cancer, lung cancer, breast cancer, liver cancer,nasopharyngeal cancer, esophageal cancer, prostate cancer, stomachcancer, cervical cancer and brain cancer.
 2. The method of claim 1,wherein said cancer or arginine-dependent tumor growth isargininosuccinate synthetase-negative.
 3. The method of claim 1, whereinthe two albumin-binding domains of the first portion are the same. 4.The method of claim 1, wherein the two albumin-binding domains of thefirst portion are different from each other.
 5. The method of claim 1,wherein said albumin-binding domain is SEQ ID NO: 46, 47, 48, or
 49. 6.The method of claim 1, wherein the linker molecule comprises a sequenceselected from SEQ ID NO: 50, 51, 52, 53, or serine-glycine-serine (SGS)amino acid sequence.
 7. The method of claim 1, wherein the argininedeiminase is selected from arginine deiminase produced from aMycoplasma, Lactococcus, Pseudomonas, Streptococcus, Escherichia,Mycobacterium or Bacillus microorganism.
 8. The method of claim 1,wherein the arginine deiminase is produced from Mycoplasma arginini,Lactococcus lactis, Bacillus licheniformis, Bacillus cereus, Mycoplasmaarthritidis, Mycoplasma hominis, Streptococcus pyogenes, Streptococcuspneumoniae, Mycobacterium tuberculosis, Pseudomonas plecoglossicida,Pseudomonas putida, Pseudomonas aeruginosa, thermophilic Aspergillusfumigatus or a combination thereof.
 9. The method of claim 1, whereinsaid weekly or biweekly administering of said albumin-binding argininedeiminase fusion protein to said patient is 1.3 mg/kg/week.
 10. Themethod of claim 1, wherein said albumin-binding arginine deiminasefusion protein is clinically effective in a pH range from 5.5 to 9.5.11. The method of claim 1, wherein said albumin-binding argininedeiminase fusion protein is clinically effective at pH 7.4.
 12. Themethod of claim 1, wherein said albumin-binding arginine deiminasefusion protein is clinically effective at pH 6.5.
 13. The method ofclaim 11, wherein said albumin-binding arginine deiminase fusion proteinhas a specific activity of about 19 U/mg at pH 7.4.
 14. The method ofclaim 1, wherein said albumin-binding arginine deiminase fusion proteinis purified from both soluble and insoluble fractions of crude proteins.